title: Distribution of lentiviral vector integration sites in mice following therapeutic gene transfer to treat β-thalassemia.
abstract: A lentiviral vector encoding β-globin flanked by insulator elements has been used to treat β-thalassemia (β-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting β-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, β-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.
authors.affiliation: Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6076, USA.
authors.forename: Keshet
authors.initials: K
authors.lastname: Ronen
authors.forename: Olivier
authors.initials: O
authors.lastname: Negre
authors.forename: Shannah
authors.initials: S
authors.lastname: Roth
authors.forename: Charlotte
authors.initials: C
authors.lastname: Colomb
authors.forename: Nirav
authors.initials: N
authors.lastname: Malani
authors.forename: Maria
authors.initials: M
authors.lastname: Denaro
authors.forename: Troy
authors.initials: T
authors.lastname: Brady
authors.forename: Floriane
authors.initials: F
authors.lastname: Fusil
authors.forename: Beatrix
authors.initials: B
authors.lastname: Gillet-Legrand
authors.forename: Kathleen
authors.initials: K
authors.lastname: Hehir
authors.forename: Yves
authors.initials: Y
authors.lastname: Beuzard
authors.forename: Philippe
authors.initials: P
authors.lastname: Leboulch
authors.forename: Julian D
authors.initials: JD
authors.lastname: Down
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Payen
authors.forename: Frederic D
authors.initials: FD
authors.lastname: Bushman
mesh_terms: D000818:Animals; D016026:Bone Marrow Transplantation; D002851:Chromatography, High Pressure Liquid; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics*; D025743:HMGA2 Protein / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D055544:beta-Globins / Q000235:genetics / Q000378:metabolism; D017086:beta-Thalassemia / Q000235:genetics / Q000378:metabolism / Q000628:therapy*
chemical_list: D025743:HMGA2 Protein; D055544:beta-Globins